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EVALUATION OF PHARMACOLOGICAL ACTIVITY OF GREWIA VILLOSA

GREWIA villosa

INTRODUCTION:

Nature has been a source of medicinal agents for thousands of years and an impressive number of modern drugs have been isolated from natural resources.

Recently, the acceptance of traditional medicine as an alternative form of health care has reinvigorated many scientists in the herbal drug development. Moreover, the increasing use of plant extracts in the food, cosmetic and pharmaceutical industries suggests that, in order to find active compounds, a systematic study of medicinal plants is very important.

Herbal medicine is the natural system of medicine that has been practiced for more than 5000 years. Ayurveda is a Sanskrit word and its meaning is “science of life or practice of longevity” this system of health care was conceived and developed by the Rishis and natural scientist through centuries of observation, discussion and medications based on trial and error.

Herbs are the major components in all indigenous preparations of traditional medicine and common element in ayurveda, homoeopathic, naturopathic and native medicinal Indian herbal medicine accentuates prevention of disease, rejuvenation of our body systems and it extends the life span and makes healthy life in balance and harmony.

From ancient time to present, people throughout the world have maintained a vast and intimate knowledge of native plants. The plant kingdom has provided an endless source of medicinal plants, first used in crude form then as herbal teas, syrups, infusions, ointments liniments, and powders.

Herbal Drug Development is an aspect of pharmacology to know how plants are used for their medicinal potential. From the point of view of drug development, the important aspects which are to be justified include the correct part of the plant which have the active chemical constituent essential for the pharmacological activity, preliminary Phytochemical screening, The selection of solvents, extraction process , and other methodology are the important aspect to evaluate the pharmacological activity of plant .

The plant itself is the source of the biggest challenge and the correct choice underpins the beginning of success.

From identification, to method and site of harvesting, the part being used, the processing, standardization, purity and the final formulation, all have to be taken into consideration.

Special attention must also be paid to standardization of the compound, using markers if possible, bio-active markers the extract should authenticated and standardized, the information is inadequate from a drug development point of view. Far more details are required to be furnished and generation of this information is crucial to further studies. This leads to successful drug development.

In this point of view, we have selected the plant of genus Grewia for the development of a successful herbal drug formulation. The genus Grewia belongs to family Tiliaceae. This genus comprising shrubs and trees and is distributed in the warmer parts of the world. Nearly 40 species of this genus are found in India some of which are well known for their medicinal property.The different parts of different species of genus Grewia are used as folk medicine in the different part of globes. Diverse bioactivity studies on different species of genus Grewia have been reported.

We have selected the species Grewia villosa as our medicinal plant for the evaluation of its pharmacological activity.

LITERATURE:

PLANT PROFILE :

Plant name : Grewia villosa

Grewia villosa

Kingdom : Plantae

Phylum : Magnoliophyta

Class : Magnoliatae

Order : Malvales

Family : Tiliaceae

Sub family : Grewioideae.

Genus : Grewia

Species : villosa

Synonyms

Grewia corylifolia,Guill. etPerrott.

Common names

English name :mallow raisin, mallow-leaved ross berry, round leaf Grewia

Sanskrit : Gudasarkara, Nagabala

Hindi : Kakarundah, Kukurbicha, Phrongli

Tamil : Kalunnu, Tavidu, Tavadu, Tavuttai

Telugu :Chimachipuru, Jibilike, Chittijana, Jibilika

Kannada  : Udipe, Dadachelu, Gandaudipe, Chikkudipe

Marathi  : Govli

Oriya : Kakarolim

Grewia villosa

Habitat and Distribution:

Grewia villosa is a very slow growing shrub of the arid areas in Africa and India, often on river banks liable to flooding or on stony ground, in the shade of larger trees. In Kenya for instance, it is found at the coast as well as in the dry south and north. In India it is found in the dry regions of Andhra Pradesh, Gujarat, Karnataka, Kerala, Madhya Pradesh, Maharashtra, Punjab, Rajasthan, Tamil Nadu.

Geographic Distribution:

Native : Angola, Botswana, Cape Verde, Eritrea, Ethiopia, India, Kenya, Mozambique, South Africa, Sudan, Swaziland, Tanzania, Uganda.

Biophysical limits:

===== Altitude: 500-1500 m

Mean annual temperature: Tolerates very high temperatures.

Mean annual rainfall: 200-800 mm

Soil type: In the Sahel, it is found on stony and

rocky; ferruginous soils, sometimes on sands along the banks of the Nile and on

periodically individual lands. =====

EXPERIMENTAL METHODOLOGY

PLANT MATERIAL

The plant material consists of dried powdered leaves, stem of Grewia villosabelonging to the family Tiliaceae.

PLANT COLLECTION AND AUTHENTIFICATION:

Grewia villosa leaves and stem were collected from A.P. FOREST ACADEMY, Bahadurpally, Quthbullapurmandal, secunderabad during the month of November. The plant is authenticated by BOTANICAL SURVEY OF INDIA, and the letter is enclosed in annexure with the voucher number STK9RGU62. Fresh leaves and stem were collected, washed and shade dried for two weeks and powdered mechanically. The dried powder was sieved through mesh #44 and stored in air tight container until the time of use.

EXTRACTION PROCEDURE :

Drug extracts preparation

Process

The drug extracts are prepared by continuous soxhlet extraction method. This method of extraction is only required where the desired compound has a limited solubility in a solvent and the impurity is insoluble in that solvent. Normally a solid material containing some of the desired compound is placed inside a thimble made from thick filter paper which is loaded into the main chamber of the soxhlet extractor. The soxhlet extractor is placed onto the round bottomed flask and a condenser is attached to it.

The solvent was heated to reflux. The solvent vapors travels up to a distillation arm and floods into the chamber. The condenser ensures that a solvent vapors cools and drips back down into the chamber housing containing the solid material of the compound. The chamber containing the solid material slowly fills with warm solvent; some of the desired compound will then dissolve in the warm solvent. When the soxhlet chamber is almost full, the chamber is automatically emptied by a siphon side arm with the solvent running back down to the distillation flask. The cycle may be allowed to repeat many times over hours or days.

During each cycle, a portion of the non-volatile compound dissolves in the solvent. After many cycles, the desired compound is concentrated in the distillation flask. The advantage of this system is that instead of many portions of warm solvent being passed through the sample, just one batch of solvent is recycled. After extraction, the solvent was removed typically by means of a rotary evaporator yielding the extracted compound. The non-soluble portion of the extracted solid remains in the thimble and is usually discarded.

Continuous extraction of leaves:

Solvents used: Methanol, Methanol, Chloroform.

Methanolic extract:

• 50gms of powdered and shade dried leaves were packed and kept for soxhlet extraction for 7 days (10cycles per day). • The yield obtained was found to be 3 gms. • The percentage yield was found to be 6%w/w.

==== Ethanolic extract: ====

• 50gms of powdered and shade dried leaves were packed and kept for soxhlet extraction for 7 days (10cycles per day). • The yield obtained was found to be 4.2gms. • The percentage yield was found to be 8.4%w/w.

Chloroform extract:

• 50gms of powdered and shade dried leaves were packed and kept for soxhlet extraction for 3 days (10cycles per day). • The yield obtained was found to be 2.15gms. • The percentage yield was found to be 4.30%w/w

Continuous extraction of stem:

Solvents used: Methanol, Methanol, Chloroform.

Methanolic extract:

• 50gms of shade dried and powdered stem were packed and kept for soxhlet extraction for 7 days (10cycles per day). • The yield obtained was found to be 1.8gms. • The percentage yield was found to be 3.6%w/w.

Ethanolic extract:

• 50gms of shade dried and powdered stems were packed and kept for soxhlet extraction for 7 days (10cycles per day). • The yield obtained was found to be 3.5gms. • The percentage yield was found to be 7%w/w.

Chloroform extract:

• 50gms shade dried and powdered stems were packed and kept for soxhlet extraction for 3 days (10cycles per day). • The yield obtained was found to be 1.7gms. • The percentage yield was found to be 3.4%w/w.

PHYTOCHEMICAL ANALYSIS OF THE EXTRACTS:

1. Detection of alkaloids:

Extracts were dissolved individually in dilute Hydrochloric acid and filtered.

a)Mayer’sTest: Filtrates weretreatedwith Mayer’s reagent (Potassium Mercuric Iodide).Formationof ayellowcoloredprecipitate indicatesthepresence of alkaloids.

B) Wagner’s Test: Filtratesweretreatedwith Wagner’s reagent (Iodine in Potassium Iodide). Formation of brown/reddish precipitate indicates the presence of alkaloids.

c) Dragendroff’sTest:Filtratesweretreated with Dragendroff’s reagent (solution of PotassiumBismuthIodide).Formation ofred precipitate indicates the presence of alkaloids.

d)Hager’sTest: Filtratesweretreatedwith Hager’s reagent (saturated picric acid solution). Formationof yellowcolored precipitate confirms the presence of alkaloids.

2. Detectionofcarbohydrates:

Extractswere dissolved individually in5 ml distilled water and filtered.The filtrateswereusedto test forthe presence of carbohydrates. a) Molisch’sTest:Filtratesweretreatedwith2 drops of alcoholic α-naphthol solution ina testtube.Formationofthevioletring atthe junction indicates the presence of Carbohydrates. b) Benedict’sTest: Filtratesweretreatedwith Benedict’sreagentandheatedgently. Orange red precipitate indicates the presence of reducingsugars. c) Fehling’s Test:Filtrates were hydrolyzed with dil. HCl, neutralized with alkali and heated with Fehling’s A & B solutions. Formation of red precipitate indicates the presence of reducing sugars.

3. Detection of glycosides:

Extracts were hydrolyzed with dil. HCl, and then subjected to test for glycosides. a)Modified Borntrager’s Test:Extracts were treated with Ferric Chloride solution and immerse in boiling water for about 5 minutes. The mixture was cooled and extracted with equal volumes of benzene. The benzene layer was separated and treated with ammonia solution. Formation of rose-pink colour in the ammonical layer indicates the presence of anthranol glycosides.

4.Legal’s Test:

Extracts were treated with sodium nitropruside in pyridine and sodium hydroxide.  Formation of pink to blood red color indicates the presence of cardiac glycosides.

5. Detection of saponins:

a)Froth Test:Extracts were diluted with distilled water to 20ml and this was shaken in a graduated cylinder for 15 minutes. Formation of 1 cm layer of foam indicates the presence of saponins. b) Foam Test:0.5gms of extract was shaken with 2 ml of water. If foam produced persists for ten minutes it indicates the presence of saponins.

6. Detection of phytosterols :

a) Salkowski’s Test: Extracts were treated with chloroform and filtered. The filtrates were treated with few drops of Conc. Sulphuric acid, shaken and allowed to stand. Appearance of golden yellow color indicates the presence of triterpenes. b)Libermann Burchard’s test:Extracts were treated with chloroform and filtered. The filtrates were treated with few drops of acetic anhydride, boiled and cooled. Concentrated sulphuric acid was added. Formation of brown ring at the junction indicates the presence of phytosterols.

7. Detection of phenols

Ferric Chloride Test:Extracts were treated with 3-4 drops of ferric chloride solution. Formation of bluish black color indicates the presence of phenols.

8. Detection of tannins

Gelatin Test:To the extract, 1% gelatin solution containing sodium chloride was added. Formation of white precipitate indicates the presence of tannins.

9. Detection of flavonoids

a)Alkaline Reagent Test:Extracts were treated with few drops of sodium hydroxide solution. Formation of intense yellow color, which becomes colorless on addition of dilute acid, indicates the presence of flavonoids. b)Lead acetate Test:Extracts were treated with few drops of lead acetate solution. Formation of yellow color precipitate indicates the presence of flavonoids.

10. Detection of proteins and amino acids:

a)Xanthoproteic Test:The extracts were treated with few drops of conc. Nitric acid. Formation of yellow color indicates the presence of proteins. b)Ninhydrin Test:To the extract, 0.25% w/v ninhydrin reagent was added and boiled for few minutes. Formation of blue color indicates the presence of amino acid.

11. Detection of diterpenes:

Copper acetate Test:Extracts were dissolved in water and treated with 3-4 drops of copper acetate solution. Formation of emerald green color indicates the presence of diterpenes[18].

ACUTE TOXICITY STUDIES:

DETERMINATION OF ACUTE TOXICITY (LD50):

The acute toxicity studies were performed to find out the LD 50 of the extracts.

Number of animals required: 6 rats (male) Number of groups: 2 groups (3 animals each group) Dose levels: 100, 300, 1000, 2000mg/kg body weight of the animals. Study duration: 3 days

Preparation of dose:

Methanolic extract of Grewia villosa leaves was suspended in 1% tweem80 solution in distilled water.

Reproductive Biology:

Flowering occurs in June-September in India whereas in South Africa fruits are ready

in the April-May period.

Botanic description:

Grewia villosa is a deciduous shrub to about 3 m with very distinctive leaves, young

parts covered with pale silky hairs (villosa).

Leaves are broadly elliptic to almost round, up to 12 cm long, 3-veined from the base, thickly textured, grey-green and hairy above with sunken veins, much paler below, covered in rough greyish-white, cream or rusty hairs; base slightly cordate; apex rounded; margin toothed.Flowers are yellow-red-brown coloured 2 cm in diameter, in small leaf-opposed clusters. Fruitsare shallowly 4-lobedand are soft and hairy when ripe, red brown coloured, about 1.5cm cross, 1-2 seeds within each nut.

Food:

The fruit may be eaten, but is not actively sought after, although it may be

found in some local markets, for instance in the Sudan, as a substitute for G. tenax.

Fodder:

The leaves are very palatable to livestock, making it a good fodder in its native range.

Timber:

The wood is made into walking sticks, bows, arrows and spear shafts.

Gum or resin:

In Sudan, an extract from the bark is used as glue for tobacco leaves.

Medicine:

Many medicinal uses are reported. In Kenya, the bark (powdered or

fresh), is used to treat wounds, and elsewhere various parts of the tree are used in the

treatment of syphilis, spleen trouble, eye-ache and stomach-ache.

Studies done on Grewia species so far:

G. serrulatashowed anti-inflammatory activity. The bark of G. tiliaefoliais used to heal wounds, cure kapha, vata, burning sensation, throatcomplaints, biliousness and disease of the nose and blood. It is alsoused in dysentery and externally employed to remove the irritationfrom cow-itch. Its wood in powder form is emetic and antidote toopium poisoning.

In G. tiliaefolia, the ethanolic extract of aerial partsexhibited CNS depressant and diuretic activity while that of stem barkexhibited spermic and hypotensive activity. The leaves of G.umbellataare used for treating cuts and wounds. The bark and roots ofGrewiatiliaefoliaare used to treat skin diseases, hypertension, ulcersand diarrhea.

Various parts of G. hirsutaare used in headache, eye complaints, sores and cholera while ethanolic extract of stem bark exhibited antiviral and diuretic activity. The leaves are useful in nose and eye diseases, treating splenic enlargement, piles, rheumatism and relieving joint pain while the roots are used in diarrhea, dysentery and as a dressing for wounds.

The plant G. microcosis used for treating indigestion, eczema and itch, small pox, typhoid fever, dysentery and syphilitic ulceration of the mouth. Grewiamollisis known to be a strong fireresistant. Various parts of the plant are used in food and medicine.

The fruit of G. asiaticais astringent and cooling. Infusion of bark is demulcent while leaves are used in pustular eruptions. Its root bark is used as a remedy for rheumatism. 50% Ethanolic extract of aerial parts of G. asiatica showed hypotensive activity while the aqueous extract of stem bark is reported to be antidiabetic. Its seed extract and seed oil exhibited anti-fertility activity. Fruit Extract of G. asiatica shows radioprotective action.

Effect in Swiss Albino Mice against Lethal Dose of γ- irradiation7.The fruit is astringent and stomachic. It is reported that unripe phalsa fruit alleviates inflammation and is administered in respiratory, cardiac and blood disorders, as well as in fever reduction. Furthermore, infusion of the bark is given as a demulcent, febrifuge, and treatment for diarrhea.

"Grewia asiatica" contains anthocyanin type cyanidin 3- glucoside9, vitamin C, minerals and dietary fibers, etc.

Pet. ether extract of G. bicoloris used for treating postulant skin lesions. Grewia bicoloris a part of Sudanese traditional medicine, and is used in the treatment of skin lesions and sometimes also as a tranquilizer. The three alkaloids, Harman, 6- methoxyharman, and 6-hydroxyharman, isolated from the methanol extract of this plant, have antibacterial properties.

Chloroform extract of the aerial parts of G. bilamellataexhibited antimalarial activity against the D6 and W2 clones of P. falciparum. Grewia carpinifoliais used in washing hair to remove and prevent lice. Ethanolic extract of stem bark of G. elastic showed CNS depressant activity.